Characterization and protective properties of attenuated mutants of Salmonella choleraesuis

We have constructed crp::Tn10 and cya::Tn10 Salmonella choleraesuis mutants and their fusaric acid-resistant derivatives with deletions (Δ) of the Tn10 and adjacent DNA sequences and found them to be avirulent and able to induce protection against a wild-type challenge in 8-week-old BALB/c mice. Mice survived infection with the crp and cya mutants at doses of more than 7 x 10³ times the oral (p.o.) 50% lethal dose (LD₅₀) and more than 8 x 10² times the intraperitoneal LD₅₀ of the wild-type S. choleraesuis parent. Mice vaccinated with attenuated strains were protected against challenge with more than 1.6 x 10⁴ times the p.o. LD₅₀ and more than 80 times the intraperitoneal LD₅₀ of the wild-type virulent S. choleraesuis parent. One deletion mutation isolated in the crp region extends to an adjacent gene(s) that was shown to be associated with avirulence. This gene or operon has been designated cdt (colonization of deep tissues). A Δ(crp-cdt)19 strain, when complemented with the wild-type crp gene and promoter on a pBR-derived plasmid, had p.o. LD₅₀ values 10³ times higher than those for the wild type. A Δcya Δ(crp-cdt)19 double mutant was less virulent than and afforded more complete protection against a challenge with the wild-type strain than a Δcrp-11 Δcya double mutant or the individual cya, crp, or crp⁺/cdt mutants. The deletion derivatives exhibited reduced invasion of CHO cells in vitro, and the numbers of the mutants recovered from mouse tissues were less than that of the parent strain. These studies suggest that one or more of the genes involved in cell attachment to and/or invasion of S. choleraesuis may be under catabolite repression. In addition, we describe a new deletion of a gene(s) located in the crp region between cysG and argD that is associated with virulence in S. choleraesuis.